The present invention relates to microbiological quality test of a sample. More specifically, the invention relates to a method for selectively detecting and/or determining the ATP from microorganism cells in a sample.
Bioluminescence analysis using luciferase is utilized as a rapid method for detecting ATP from microorganism cells. As a method for detecting the ATP from microorganism cells in a sample that is a mixture of microorganism cells and non-microorganism cells, a method is employed which comprises dissolving non-microorganism cells with a nonionic surface active agent, decomposing the released ATP from the non-microorganism cells with an ATP hydrolase such as apyrase, inactivating the apyrase with, for example, boiling-Tris buffer, adding an agent for lysing microorganisms to the sample and quantitatively determining the ATP from the microorganism cells by bioluminescence analysis (U.S. Pat. No. 3,745,090). According to this method, test results can be obtained in only several ten minutes and, thus, this method is most appropriate for sampling inspection and the like. However, since the inactivation treatment of apyrase with boiling-Tris buffer causes a decrease in determination sensitivity, it has not been possible to obtain a correct value for the ATP level.
As a method for selectively determining the ATP from microorganism cells in a sample containing non-microorganism cells, the three methods described below have been developed (Japanese Examined Patent Publication No. 62-4120). In any of these methods, however, selective detection with a high sensitivity for the ATP from microorganism cells in the presence of the abundant ATP from non-microorganism cells is performed with much difficulty. The first method comprises treating a sample with a nonionic surface active agent to extract the ATP from non-microorganism cells, separating the ATP through a filter, extracting the concentrated ATP from microorganism cells in a filter and determining this ATP by bioluminescence analysis. This method, however, is not an appropriate method since it requires a long time and complicated operations for the filtration. The second method comprises treating a sample with a nonionic surface active agent to extract the ATP from non-microorganism cells, centrifuging the sample to remove the above ATP as a supernatant, extracting the ATP from microorganism cells from the precipitate containing microorganisms and determining this ATP by bioluminescence analysis. The drawback of this method is that the ATP from non-microorganism cells cannot be perfectly separated through one centrifugal operation. Although perfect separation is achieved by repeating centrifugal operations, these operations require a long time and are complicated. Furthermore, as the number of washing cycles (consisting of centrifugation and removal of supernatant) increases, the yield of microorganism cells decreases. Thus, this method cannot be said a good one. The third method comprises treating a sample with a nonionic surface active agent or the like to extract the ATP from non-microorganism cells, decomposing this ATP with an ATP hydrolase such as apyrase, passivating the apyrase with glass beads or the like after completion of the decomposition, then extracting the ATP from microorganism cells and determining this ATP by bioluminescence analysis. In this method, however, highly sensitive detection cannot be expected because the sample does not undergo an operation to concentrate microorganism cells and because the method is susceptible to determination inhibition by components of the sample such as proteins and milk fat. Furthermore, in this method, the removal or deactivation of the apyrase is incomplete and the inhibition action of the apyrase persists even after the extraction of ATP from microorganism cells. Thus, a correct ATP level cannot be determined. Accordingly, ATP values determined by the above three methods vary widely and, actually, the detection sensitivity that can be expected is only about 10.sup.6 CFU/ml (CFU: colony forming unit).
As one of the promising methods for improving detection sensitivity in bioluminescence analysis of ATP, the improvement of the above-described method including centrifugal operations in which an increase in sensitivity by concentration of microorganism can be expected may be mentioned. For milk samples, in particular, a new method has been proposed in which a chelating agent and a nonionic surface active agent are added to milk and then centrifuged in order to separate and concentrate microorganism cells from other components that inhibit the ATP determination. According to this method, determination inhibition by other milk components can be prevented. However, this method requires complicated operations, and an increase in detection sensitivity is hardly recognized since the removal of the ATP from non-microorganism cells is insufficient. Thus, as a practical testing method, insufficiency in sensitivity cannot be denied (EP-A-542790).
In view of these facts, the present invention aims at providing a method for selectively detecting and/or determining the ATP from microorganism cells in a sample at a high sensitivity and in a short time.